Heparin is the injectable anticoagulant of choice for surgical patients requiring either vascular surgery or therapeutic intervention for cardiovascular disease, as well as for the post-surgical management of an immobile (e.g., orthopedic) patient. For example, it is administered to almost all patients during cardiac catheterization, angioplasty, cardiopulmonary bypass and the treatment of unstable angina and myocardial infarction (Bauer, T. L., et al., Circulation, 95: 1242–1246 (1997)). However, the therapeutic use of heparin is associated with some serious side effects including the development of heparin-induced thrombocytopenia (Chong, B. H., et al., Blut, 58:53–57 (1989)).
Two types of heparin-induced thrombocytopenia with distinct etiologies are recognized. Type 1 is characterized by a relatively mild thrombocytopenia of early onset, that is, within the first few days of heparin therapy. It usually resolves without cessation of heparin (Jackson, M. R., et al., Surgery 121:419–424 (1997)). This type of thrombocytopenia is thought to be caused by an intrinsic proaggregatory effect of heparin (Chong, B. H., et al., Eur. J. Haematol., 43:245–251 (1989)). Clinical complications of type 1 thrombocytopenia are uncommon. Type 2 heparin-induced thrombocytopenia (referred to herein as HIT), is a drug-induced, immunoglobulin-mediated thrombocytopenic disorder that is often associated with severe venous or arterial thrombosis attributed to the formation of platelet thrombi (Cines, D. B., et al., N. Engl J Med, 31:581 (1987); Celoria, G. M., et al., Angiology, 39:915 (1988)). HIT generally occurs 5–8 days following the first exposure to heparin. Platelet counts often drop to less than 100×109/1 and in rare cases to 20×109/1. The incidence of HIT is variable, with rates of occurrences as high as 24–30% reported in some of the earlier studies (Nelson, J. D., et al., Arch. Intern. Med., 138:548–552 (1978); Bell, W. R., et al., Ann. Intern. Med., 85:155–160 (1976)) and with lower values (5–10%) in the more recent studies (Almeida, J. I., et al., J. Vasc. Surg., 27:309–316 (1998); Griffiths, E., and Dzik, W. H., Trans. Med., 7:1–11 (1997); Aster, R. H., N. Eng. J. Med., 332:1374–1376 (1995)). Overall, about 5% of patients receiving heparin develop HIT and about 5–80% of those develop thrombosis, which can involve the arterial, the venous, or both systems (Gupta, A. K., et al., An. Pharmacol., 32:55–59 (1998); Warkentin, T. E., et al., Am. J. Med., 101:502–507 (1996); Chong, B. H., et a., Br. J. Haematol, 89:431–439 (1995)). Patients may develop cerebrovascular complications, myocardial infarction, limb ischemia, or deep venous thrombosis. The thrombotic complications are fatal in about 29% of patients, and an additional 21% have limb amputations (King, D. J., et al., Ann. Intern. Med., 100:535–540 (1984)).
It is now widely accepted that immune-mediated (Cancio, L. C., et al, J. Am. Coll. Surg., 186:76–91 (1998); Warkentin, T. E., et al., Thromb. Haemost., 79:1–7 (1998)), and it has been demonstrated that HIT patients produce immunoglobulins reactive with a platelet/heparin complex which, in the presence of pharmacological concentrations of heparin and certain releasable platelet proteins, mediate platelet activation and aggregation, resulting in thrombosis and endothelial cell injury. Because of the underlying immunological mechanism, HIT may occur in patients receiving any preparation of heparin, at any dose, and by any route, including the low heparin concentrations required to maintain the patency of arterial lines (Johnson, R. A., et al., Am. J. Hematol., 17:349–353 (1984)). Any patient who receives heparin is at risk for type 2 heparin-induced thrombocytopenia. No known patient characteristics predict the development of this syndrome. Although an initial sensitization to heparin requires 5–14 days of heparin therapy, thrombosis and thrombocytopenia can occur on the first day of reexposure in a patient previously sensitized to heparin. The time interval required for the manifestation of symptoms can be only hours from the time of heparin reexposure. Consistent with these observations, platelet counts should be obtained before the initiation of heparin therapy and daily thereafter during the course of heparin administration.
The pathophysiology of HIT involves platelet cell surface receptors, and possibly other cell membranes (e.g., endothelial cell membranes bearing platelet factor 4) which bind particular macromolecular heparin/heparin binding protein complexes. For example, it is well established that plasma from HIT patients contains immunoglobulin reactive with a multimolecular complex comprising heparin and platelet factor 4 (PF4). Platelet factor 4 is an abundant tetrameric heparin-binding protein which is stored in platelet α-granules and released by activated platelets. Similarly, thrombospondin-1 (TSP-1) is a 420 kDa glycoprotein present in platelet a granules which accounts for 25% of the total protein secreted by platelets and which contains heparin-binding sequences in its amino terminal globular domain.
HIT-dependent platelet activation is thought to be mediated by the activation of platelet FcγRIIa receptors resulting from the binding of the Fc portion of the IgG component of an antibody/heparin/heparin binding protein complex, which ultimately is deposited onto the platelet surface (Griffiths, E., and Dzik, W. H., Trans. Med., 7: 1–11 (1997); Warkentin, T. E., Drug Safety, 17:325–341 (1997)). Furthermore, it has recently been appreciated that HIT IgG is a potent platelet agonist which is able to generate platelet procoagulant activity even more potently than classic platelet agonists (e.g., thrombin and collagen) (Warkentin, T. E., Drug Safety, 5:325–341 (1997)). Thus, the procoagulant effects of HIT antibodies and resulting endothelium activation/injury partially explain the high incidence of venous thromboembolism observed in HIT patients.
Early diagnosis of HIT is required to prevent the life-threatening complications that can occur if heparin is continuously administered to a patient producing heparin induced antibodies. It is estimated that the prompt detection of platelet/heparin complex reactive immunoglobulin, and the immediate cessation of heparin administration could reduce the morbidity rate to 7.4–23% and the mortality rate to 1.1–12% (Almeida, J. I., et al., J. Vasc. Surg., 27:309–316 (1998); Laster, J. D., et al., Surgery, 102:763–770 (1987)). HIT should be diagnosed on the basis of two criteria: one or more clinical events associated with syndrome (primarily thrombocytopenia), and laboratory evidence for a heparin-dependent immunoglobulin using a sensitive and specific diagnostic assay. Currently, diagnosis of HIT is usually made on a clinical basis and is subsequently confirmed by a positive laboratory test. However, difficulties in diagnosing HIT are common, due primarily to the fact that the laboratory tests which are currently available often produce both false negative and false positive results (Griffiths, E., and Dzik, W. H., Trans. Med., 7:1–11 (1997); Newman, P. M., et al., Thromb. Haemost., 80:292–297 (1998)).
Two standard tests based on platelet aggregation or activation include the platelet aggregation test (PAT) and the 14C-serotonin release assay (SRA). Currently, the PAT is the most commonly performed laboratory test for diagnosis of HIT. This assay has the advantage of being a non-radioactive test and employs a standard methodology. However, it is considered to be specific but not sensitive, with a false negative rate as high as 50% (Greinacher, A., et al., Thromb. Haemost., 66:734–251 (1991)). The SRA is sensitive and specific and is the recommended assay for HIT (Visentin, G. P., et al., J. Clin. Invest., 93:81–88 (1994); Kelton, J. G., et al., Blood, 72: 925–930 (1998); Sheridan, D. C., et al., Blood, 67:27–30 (1986)). However this assay employs radiolabeled serotonin and considerable expertise is required to obtain reproducible and reliable results (Kelton, J. G., et al., Blood, 72:925–930 (1988); Sheridan, D. C., et al., Blood, 67:27–30 (1986)). The reported sensitivity of the PAT has varied from 17% to 81% and that of the SRA from 29% to 94% depending on the study (Cancio, L. C., and Cohen, D. J., J. Am. Coll. Surg., 186:76–91(1998)), a consideration which limits the diagnostic usefulness of both of these standard tests.
Recently, an enzyme-linked immunosorbent assay (ELISA) using a complex of recombinant platelet factor 4 (rPF4) and heparin as a target was developed (Amiral, J. F., et al., Thromb. Haemost., 73:21–28 (1995); Amiral, J. F., et al., Thromb. Haemost., 68:95–96 (1995)). Several recent studies have compared the sensitivity of the rPF4/heparin ELISA with PAT or SRA. Sera from 209 patients were tested in one study (Greinacher, A. J., et al., Transfusion, 34:381–385 (1994)) that compared the rPF4/heparin ELISA with the PAT. The ELISA was positive in 33% and the PAT in 11.5% of cases. The improved sensitivity of the rPF4/heparin ELISA has been partly attributed to its ability to detect IgM and IgA antibodies in addition to IgG. The rPF4/heparin ELISA is now commercially available. The rPF4/heparin ELISA uses non-radioactive reagents. However, it produces a high background and is considered to be relatively insensitive (Newman, P. M., et al., Thromb. Haemost., 80:292–297 (1995)).
Published studies evidence the fact that 10 to 50% of test samples evaluated in both a functional assay (e.g., PAT or SRA) and antigen (rPF4 ELISA) assay generate discordant results (Newman, P. M., et al., Thromb. Haemost., 80:292–297 (1998); Greinacher, A. J., et al., Transfusion, 34:381–385 (1994); Arepally, G. C., et al., Am. J. Clin. Path., 104:648–654 (1995)). More recently, a multicenter clinical trial of the thrombin inhibitor, argatroban, was conducted in patients with HIT and patients with HIT that had progressed to thrombosis (Walenga, J. M., et al., Sem. in Hematology, 36:22–28 (1999)). In this study, three HIT diagnostic assays were investigated: the PAT, the SRA and the ELISA for the antibody to PF4/heparin complex. Confirmation was made in 26%, 55%, and 64% of the patients, respectively (n=199 patients) (Walenga, J. M., et al., Sem. in Hematology, 36:22–28 (1999)). Combined results of the three assays enhanced the positive response to 83% of the total population. These data demonstrate that there is no direct correlation between the positive response of these assays, and that clinically positive HIT patients can be missed by all three standard assays.
Thus, there is a need for a sensitive and specific diagnostic assay for the early detection of heparin/heparin binding protein complex-reactive immunoglobulin in the plasma of patients receiving heparin therapy.